1. Role of Membrane Interactions on the Mechanism of alpha-Synuclein Amyloid Formation Understanding the environmental factors affecting the aggregation of alpha-synuclein (alpha-syn) is of great importance because the accumulation and deposit of alpha-syn are intimately connected to Parkinsons disease (PD) etiology. Membrane interactions are of particular interest because alpha-syn localizes near synaptic vesicles and mitochondrial membranes in vivo. Specifically, the protein undergoes disordered-to-helical structural changes with the addition of membrane mimics such as SDS micelles and upon binding to anionic phospholipid vesicles (SUV) of varying size and composition. To develop a detailed understanding of how membranes influence alpha-syn conformation, site-specific probes of protein conformational heterogeneity and polypeptide-membrane interactions are necessary. Fluorescence spectroscopy is particularly suited for this application because of the availability of environmentally sensitive fluorophores and the ease of performing experiments near physiological temperatures and concentrations even down to a single molecule. In our studies, we have employed anionic SUVs and SDS micelles as membrane mimics to investigate membrane-induced conformational changes by fluorescence as well as circular dichroism (CD) spectroscopy. Tryptophan was substituted at four different aromatic residues (F4W, Y39W, F94W, and Y125W) to report information on local polypeptide environment and conformational heterogeneity between SUV- and SDS-micelles-bound alpha-syn. With this approach, we aim to determine the crucial protein-to-membrane conditions and key sites of interaction that promote protein aggregation and ultimately, monitor membrane-mediated amyloid formation processes. 1.1 Tryptophan Probes at the alpha-Synuclein Membrane Interface Measurements of steady-state and time-resolved fluorescence of single tryptophan-containing alpha-syn variants have revealed distinct phospholipid vesicle and micelle interactions at residues 4, 39, 94, and 125 (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622). Our circular dichroism (CD) data confirm that Trp mutations do not affect alpha-syn membrane binding properties saturating at an estimated lipid-to-protein molar ratio of 380 or approximately 120 proteins covering 7% of the surface area of an 80 nm diameter vesicle. Fluorophores at positions 4 and 94 are the most sensitive to the lipid bilayer with pronounced spectral blue-shifts (W4: lambda max 23 nm;W94: lambda max 10 nm) and quantum yield increases (W4, W94: 3 fold) while W39 and W125 remain primarily water-exposed. Time-resolved fluorescence data show that all sites (except W125) have subpopulations that interact with the membrane. Specifically, the presence of protein conformational heterogeneity in the bilayer, suggest that the both W4 and W94 exhibit high membrane affinity. Notably, both of these sites have not been characterized previously in the vesicle-bound alpha-syn structure. 1.2 Promotion of Inter-alpha-Synuclein Interactions by the Membrane Surface There is a strong relationship between membranes and alpha-syn aggregation behavior, measurements of protein conformation and dynamics on the membrane surface are necessary to gain insight into how this protein converts from a benign to a pathogenic form. We have begun to investigate the specific relationship between amyloid formation and vesicle concentration. Initial data indicate that under low lipid-to-protein ratios (<140, below the midpoint transition) protein aggregation is stimulated with the lag phase times hasten from protein alone or under saturation conditions. This is striking because using our equilibrium isotherm data (Pfefferkorn CM, Lee JC, J. Phys. Chem. B 2010, 114, 4615-4622), we would estimate a staggering 14,000 proteins-per-vesicle (lipid-to-protein 4);a value significantly higher than the maximum binding sites per vesicle (120). Under these solution conditions, it is not possible for all proteins to adopt known membrane-bound structures. While our data suggest that alpha-syn proteins are not closely packed at saturation, it is plausible that changing the lipid-to-protein ratio could result in conformational rearrangement at the vesicle surface. The local conformational change between the solvent-to-lipid or different lipid-bound states is evidenced in the lipid concentration dependent W94 time-resolved data, indicating that protein-membrane stoichiometry is crucial in modulating membrane interactions in this region of the protein. Because all W4 populations are sensitive to the membrane at all lipid concentrations, while W94 has both lipid and solvent exposed conformers that are dependent on lipid-to-protein ratio, we suggest that W4 could act as a membrane anchor. It is noteworthy that the hydrophobic non-amyloid beta component (NAC, 61-95) region has been proposed to be secluded from intermolecular contacts by the intramolecular N- and C-terminal interactions. Similarly, it is feasible for membrane stimulated aggregation to arise because as the local protein concentration increases at the surface, polypeptide conformational rearrangement ensues leading to exposure of potential protein-protein interaction sites promoting amyloid formation. Currently, experiments using bimolecular quenching and Forster energy transfer measurements are underway to identify and define conformational changes that ensue under these conditions that promote protein aggregation. 2. Fluorescent Probes of alpha-Synuclein Fibril Assembly Upon aggregation, alpha-syn undergoes conformational changes from a disordered monomer to beta-sheet fibrils (amyloid). Importantly, amyloids have been shown as the major constituent in Lewy bodies, a pathological hallmark of PD. A mature fibril is approximately 5&#8722;20 nm in diameter with varying lengths (up to micrometers) and can be comprised of single or multiple filaments (2&#8722;5 nm). These filamentous structures are characterized by a cross-beta fold where each beta-strand is aligned perpendicular to the fibril axis with interstrand hydrogen bonds running along the fibril. Though it is generally thought that the alpha-syn fibril core is composed of residues 30&#8722;100, the molecular details of fibrillar assembly is poorly understood especially pertaining to the N- and C-terminal regions. We have prepared single-Cys mutants derivatized with an environment sensitive dansyl (Dns) fluorophore at specific residues spanning across the alpha-syn sequence (G7C, V26C, V51C, V66C, V77C, L100C, and Y136C) and characterized both soluble and aggregated forms by measurements of steady-state and time-resolved fluorescence. To unravel the role of these individual residues, we utilized these fluorescent variants to monitor the fibril assembly kinetics of the wild-type protein (1.5 Dns-protein: 100 wild-type). Dns fluorescence demonstrates to be generally an effective probe, particularly, changes in both mean emission wavelength and quantum yield are sensitive to amyloid formation.